In the DNA of many organisms, including mammals, a fraction of the cytosines become methylated after DNA replication. Although a link has been demonstrated between methylation and inactivity of DNA, the function and mechanism of DNA methylation remains unclear.In particular, almost nothing is known about what determines which sequences become methylated. Features of the relatively simple eukaryote Neospora crassa will allow progress on this and other methylation problems that have been refractory to analysis in higher eukaryotes. In Neurospora, DNA sequences that are normally methylated are faithfully methylated de novo after they are reintroduced into the organism by transformation. This, together with the ability to induce methylation of selected chromosomal regions by RIP (repeat-induced point mutation) opens the way to determine experimentally what triggers methylation. Finally, classical genetic techniques for Neurospora will facilitate both mechanistic and functional studies of DNA methylation. Analysis of sequences altered by RIP has indicated that a sprinkling of G:C to A:T mutations can convert a normally unmethylated sequence into a methylated region. The planned experiments should elucidate the causal relationship between the mutations and methylation, test a model, and provide basic information essential to reach a firm understanding of DNA methylation in eukaryotes. A combination of in vivo and in vitro approaches will reveal which sites must be mutated (and whether various mutations are equivalent) to trigger methylation of a chromosomal region. According to the "collapsed chromatin" model, chromosomal regions that lack bound non-histone chromosomal proteins over a certain span condense spontaneously, and secondarily become methylated. Specific experiments will test'if a normally methylated region (zeta-eta) can be kept unmethylated by placing it in an active gene (am) or next to a site bound by a nonhistone chromosomal protein (qa-1F). The general idea that methylation is the default state will be addressed by looking for short (ie. <50 bp) sequences capable of preventing or inducing methylation of surrounding sequences. Mutants will be isolated and employed to investigate the function of methylation, and to further explore mechanism. To test whether DNA methylation is essential for viability or normal development of N. crassa, the DNA methyltransferase gene of N.crassa will be isolated and inactivated by RIP and/or "gene replacement". Mutants defective in other genes necessary for methylation will be isolated using two approaches - one using a selection successful in a preliminary experiment, and the other using "brute force" to obtain all possible mutants.